Project Summary Despite the clear importance of IFN-? for control of M. tuberculosis infection, there are still fundamental gaps in understanding mechanisms of IFN-? dependent immunity to this globally significant pathogen. An important long-term goal is to understand whether and how IFN-? induces macrophages to restrict nutrients from the M. tuberculosis containing phagosome. Lipids, including triglycerides and cholesterol, are important nutrient sources for M. tuberculosis during infection. In mammalian cells, lipids are often stored in lipid droplets, cytosolic organelles that consist primarily of triglycerides and cholesterol surrounded by a phospholipid monolayer. Lipid droplets are a characteristic of M. tuberculosis infected macrophages and it is thought that the bacteria exploit lipid droplets as a nutrient rich reservoir. However, lipid droplets serve a variety of biological functions, and can play an important role in host immune responses. Importantly, few if any studies of the role of lipid droplets during M. tuberculosis infection of macrophages have been conducted in the context of IFN-? activation. The primary hypothesis of this proposal, guided by strong preliminary data, is that in the context of IFN-? activation, lipid droplets serve to amplify the production of lipid derived eicosaonids that are essential for productive immune responses to infection, and that these lipid droplets are no longer accessible to M. tuberculosis as a nutrient source. To test this hypothesis and clarify the role of lipid droplets during M. tuberculosis infection two specific aims are proposed. 1) Elucidate the lipid droplet proteome in Mtb infected IFN-? activated macrophages and 2) Develop genetic tools for manipulating LD abundance in macrophages. Lipid droplets are known to have dynamic proteomes that mediate their cellular functions. In the first aim directed and unbiased methods to identify proteins that localize to lipid droplets will be employed to elucidate the lipid droplet proteome. Definition of cell type specific LD proteomes can provide significant clues as to context specific lipid droplet function. To maximize the sensitivity and specificity of proteomic studies, a novel proximity base biotinylation strategy will be employed. In the second aim, Cas9 mediated genome engineering will be used to specifically manipulate the abundance of LDs during infection with M. tuberculosis. The impact of decreasing LD accumulation in IFN-? activated macrophages on M. tuberculosis restriction, bacterial nutrient acquisition, and host eicosanoid production will be determined. If successful, the proposed experiments will shift the paradigm of the role of lipid droplets in M. tuberculosis pathogenesis and will uncover new mechanisms of IFN-? dependent immunity. Such knowledge will illuminate our understanding of the basis of successful immune responses to M. tuberculosis, and may inform the development of immune-modulating therapies and vaccines.